Solution 1H NMR Characterization of the Axial Bonding of the Two His

Sep 7, 2006 - The anisotropy of the paramagnetic susceptibility tensor allowed the quantitative factoring of the hyperfine shifts for the two axial Hi...
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Solution 1H NMR Characterization of the Axial Bonding of the Two His in Oxidized Human Cytoglobin Vasyl Bondarenko,† Sylvia Dewilde,‡ Luc Moens,‡ and Gerd N. La Mar*,† Contribution from the Department of Chemistry, UniVersity of California, DaVis, California 95616 and Department of Biomedical Sciences, UniVersity of Belgium, UniVersiteitsplein 1, B-2610 Wilrijk (Anterwerpen), Belgium Received May 12, 2006; E-mail: [email protected]

Abstract: Solution 1H NMR spectroscopy has been used to determine the relative strengths (covalency) of the two axial His-Fe bonds in paramagnetic, S ) 1/2, human met-cytoglobin. The sequence specific assignments of crucial portions of the proximal and distal helices, together with the magnitude of hyperfine shifts and paramagnetic relaxation, establish that His81 and His113, at the canonical positions E7 and F8 in the myoglobin fold, respectively, are ligated to the iron. The characterized complex (∼90%) in solution has protohemin oriented as in crystals, with the remaining ∼10% exhibiting the hemin orientation rotated 180° about the R-, γ-meso axis. No evidence could be obtained for any five-coordinate complex (90%) molecular species. There are several other weak peaks detected from a clearly minor component, whose intensities represent the likely methyls for a j10% minor component. Several of the resolved, low-field, single proton peaks are strongly relaxed, as is obvious in the WEFT trace in Figure 2B. Three very broad and strongly relaxed linewidths ∼500 Hz (T1 ≈ 3 ms) are observed, of which the two upfield peaks at -5 ppm (marked Y in Figure 2B) and at -9.5 ppm (labeled H81) exhibit intensities consistent with arising from one proton each, while the partially resolved lowfield peak at ∼15 ppm (marked X in Figure 2B) has an intensity indicative of more than one proton. Such broad and strongly (45) Neal, S.; Nip, A. M.; Zhang, H.; Wishart, D. S. J. Biomol. NMR 2003, 26, 215-240. (46) Cross, K. J.; Wright, P. E. J. Magn. Reson. 1985, 64, 220-231.

relaxed protons must arise from the four nonlabile ring protons of the two ligated His.44 Several low-field resonances exhibit T1 e 50 ms, of which that at 18.1 ppm (T1 ∼20 ms), among others in the window 9-20 ppm, is lost in the spectrum in 2H2O (Figure 2C). The T1 values for resolved signals are listed in Tables 1 and 2. Assignment of the Heme. TOCSY spectra (not shown; see Supporting Information) locate one CH-CH2, one CH2-CH2, and one vinyl group with significant temperature dependence to the shifts to indicate origins as heme substituents. The fourth proton to the CH-CH2 fragment (as well as the protons of the other vinyl group) is outside the spin-lock field of the TOCSY spectrum, but an intense NOESY crosspeak indicative of geminal protons (Figure 3D) and characteristic low-field resolved CRH to an upfield resolved CβH2 of a vinyl (Figure 3A) identify the four non-methyl heme substituents. Two of the three low-field resolved methyls exhibit the weak intermethyl NOESY crosspeak of 1-CH3 and 8-CH3 (Figure 3E), and NOESY contact of the former with a vinyl (Figure 3A) and the latter with a CH2-CH2 (Figure 3C) fragment identify the 2-vinyl (and hence 4-vinyl) and 7-propionate (and hence 6-propionate) heme signals. NOESY cross peaks of a CH2CH2 fragment to the remaining resolved methyl peaks at ∼36 ppm (not shown) identify 5-CH3. The remaining TOCSYdetected 4-vinyl group exhibits NOESY cross peaks to a nonresolved methyl group with no scalar connectivity (not shown) and with characteristic anti-Curie behavior (see Supporting Information) to provide the remaining 3-CH3 resonance J. AM. CHEM. SOC.

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Table 1. Observed Chemical Shifts, Dipolar Shifts, and Contact Shifts for the Heme and Two Axial His Ligands in Human Metcytoglobin δDSS(obsd)a(T1)b

δDSS(dia)c

3.6 ( 0.2 3.8 ( 0.2 2.5 ( 0.3 3.6 ( 0.2

1-CH3 3-CH3 5-CH3 8-CH3 6-CRH’s 6-CβH’s 7-CRH’s 7-CβH’s 2HR 2Hβ’s 4HR 4Hβ’s R-meso-Hh β-meso-Hh γ-meso-Hh δ-meso-Hh

18.81(105) 6.59 36.43 (95) 13.29 (105) 17.61(90), 9.68 3.41, 1.92 2.05, 2.05 -0.92, -0.07 16.23 (85) -4.76, -5.67(150) 5.22 -0.23, -0.71 2.86 1.50 5.37 3.98

NH C RH Cβ1H Cβ2H C H Cδ H

10.06 7.88 8.65 14.24(49) -8.7 (∼3) 15(2(∼3)

7.8 ( 0.4 2.7 ( 0.2 1.9 ( 0.3 1.8 ( 0.3 2.3 ( 1.0 0.0 ( 0.7

NH C RH Cβ1H Cβ2H C H Cδ H NδH

12.31 8.10 8.10 15.2(47) -4.95(∼3) 15(2(∼3) 18.4(20)

7.1 ( 0.4 2.2 ( 0.2 1.5 ( 0.3 1.7 ( 0.3 0.8 ( 0.8 -0.8 ( 0.7 7.4 ( 0.4

9.9(0.5 9.3 ( 0.5 10.2 ( 0.4 9.9 ( 0.4

δhfd

δdip(calcd)e

δconf

δdip(calcd)g

δconf

heme 15.2 ( 0.2 2.8 ( 0.2 33.9 ( 0.3 9.7 ( 0.2

-2.5 ( 0.2 -4.7 ( 0.4 -2.5 ( 0.2 -4.3 ( 0.4

17.7 ( 0.4 7.5 ( 0.6 36.4 ( 0.5 14.0 ( 0.6

-2.9 ( 0.3 -5.0 ( 0.5 -2.9 ( 0.3 -5.2 ( 0.5

18.1 ( 0.5 7.8 ( 0.7 36.8 ( 0.6 14.9 ( 0.7

-7.0 ( 0.5 -7.8 ( 0.5 -4.8 ( 0.4 -5.9 ( 0.4

-9.8(0.8 -9.6 ( 0.8 -9.8 ( 0.8 -9.6 ( 0.8

2.8 ( 1.3 1.8 ( 1.3 5.0 ( 1.2 3.7 ( 1.2

-9.9 ( 0.8 -10.9 ( 0.9 -9.9 ( 0.8 -10.9 ( 0.9

2.9 ( 1.3 3.1 ( 1.4 5.1 ( 1.2 5.0 ( 1.3

His81(E7)i[E6]k 2.8 ( 0.4 2.4 ( 0.2 5.2 ( 0.2 4.6 ( 0.4 6.8 ( 0.3 6.7 ( 0.6 12.4 ( 0.3 6.1 ( 0.5 -11 ( 1.0 19.6 ( 1.6 15 ( 2.7 14.3 ( 1.2

0.4 ( 0.6 0.6 ( 0.6 0.1 ( 0.9 6.3 ( 0.8 -30.7 ( 2.6 -0.7 ( 3.9

2.4 ( 0.2 4.2 ( 0.4 6.2 ( 0.5 6.3 ( 0.5 15.2 ( 1.3 19.9 ( 1.6

0.4 ( 0.6 1.0 ( 0.6 0.6 ( 0.8 6.0 ( 0.8 -26 ( 2.3 -4.9 ( 4.3

His113(F8)i[F15]j 5.9 ( 0.4 5.2 ( 0.4 5.7 ( 0.2 6.2 ( 0.5 7.2 ( 0.3 6.2 ( (0.5 14.4 ( 0.3 6.9 ( 0.6 -5.8 ( 0.8 17.6 ( 1.4 15.8 ( 2.7 15.7 ( 1.3 11.0 ( 0.4 12.2 ( 1.0

0.7 ( 0.8 -0.5 ( 0.7 1.0 ( 0.8 7.5 ( 0.9 -23.4 ( 2.2 0.1 ( 4.0 1.2 ( 1.4

6.3 ( 0.5 6.8 ( 0.6 6.5 ( 0.6 7.5 ( 0.6 22.2 ( 1.8 11.5 ( 1.0 14.2 ( 1.2

-0.4 ( 0.9 -1.1 ( 0.8 0.7 ( 0.9 6.9 ( 0.9 -28.0 ( 2.6 4.3 ( 3.7 -3.2 ( 1.6

a Observed chemical shift, in ppm, from DSS, in 1H O, 50 mM in phosphate, pH 7.5 at 30 °C. b T , in ms, for resolved resonances. c Chemical shift for 2 1 an isostructural diamagnetic complex, as provided by the shift X45 and the porphyrin ring current46 program. For the heme methyls and meso-protons of the 47 d cytoglobin the chemical shifts for MbCO were used. Experimental hyperfine shift (eq 1), as obtained from δDSS(obsd) - δDSS(dia). e Dipolar shift, as calculated by the optimized magnetic axes described in Table 3 and Figure 7. Magnetic axes were obtained using WT metCygb crystal coordinates.12 f Contact shift, as obtained from eqs 1 and 2. g Dipolar shift, as calculated by the optimized magnetic axes described in Table 3 and Figure 7. Magnetic axes were obtained using C38S/C83S metCygb crystal coordinates for chain B.11 h All the meso protons were artificially placed with the same distance from the Fe ion when their dipolar shift was calculated. i Helical/loop position in mammalian (i.e., axial His is F8, distal is E7, etc.) Mb fold is given in parentheses. j Actual helical/loop positions in Cygb are given in square brackets.

position. NOESY cross peaks from the substituents adjacent to a meso position to relaxed peaks, with characteristic strongly low-field intercepts (∼15-20 ppm) in a Curie plot, locate the four meso-H’s. The chemical shifts for the heme resonances are listed in Table 1. Sequence-Specific Assignment of the Proximal and Distal Helices. We use the helical/loop position of residues in the standard Mb fold as described by sperm whale Mb2,3 since this provides direct comparison of the pattern of dipolar shifts for the similarly placed residue relative to the heme. At least the peptide NH and one CβH of a ligated His can be expected to yield resonances resolved to the low-field of 10 ppm.34 Two such CβH protons are resolved in the low-field trace, and their T1 ≈ 50 ms dictates that they are significantly closer to the iron than a heme methyl (T1 ∼100 ms) and, hence, originate from coordinated His. TOCSY spectra reveal protons of two such low-field NHCRH-CβH fragments, as illustrated in Figure 4 (the CRH and CβH of one His are essentially degenerate). The second CβH is not detected in TOCSY spectra because of insufficient power in the spin lock field but is very readily detected in the NOESY spectrum by the intense cross peak indicative of geminal protons (Figure 3D). The TOCSY/ NOESY connectivities and chemical shifts are very similar to those observed for the two His in metNgb.48 12992 J. AM. CHEM. SOC.

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Six helical fragments are detected by their characteristic49 Ni-Ni+1, Ri-Ni+1, βi-Ni+1, Ri-Ni+3, and/or Ri-βi+3 NOESY cross peaks among TOCSY detected spin systems. The NiNi+1 and Ri-Ni+1 connections for the fragments of the proximal and distal helices are illustrated in Figure 5. One fragment is represented by Vali-Glyi+1-Zi+2-Alai+3-AMXi+4-Alai+5-Leui+6 (z is >4 spins) (connections are summarized in Figure 6), with one of the His backbones presented above as AMXi+4. The sequence identifies this uniquely as Val109-Leu115 and residues F4-F10 of the proximal or F helix. Hence the hyperfine shifted backbone of His113(F8) is assigned. The side chains exhibit all expected NOESY cross peaks to the heme, as shown schematically in Figure 1B. In addition to His113(F8), significant hyperfine shifts are observed for Val109(F4), Gly110(F5), Ala112(F7), and Ala114(F9) (see Table 2). The NOESY spectrum exhibits a cross peak of the His113(F8) CβH’s and NH to a moderately relaxed (T1 ∼20 ms), labile proton at 18.4 ppm (Figure 3F) which identifies the His ring NδH. Chemical shifts for the axial His are listed in Table 1, and those for the (47) The´riault, Y.; Pochapsky, T. C.; Dalvit, C.; Chiu, M. L.; Sligar, S. G.; Wright, P. E. J. Biomol. NMR. 1994, 4, 491-504. (48) Du, W.; Syvitski, R. T.; Dewilde, S.; Moens, L.; La Mar, G. N. J. Am. Chem. Soc. 2003, 125, 8080-8081. (49) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley & Sons: New York, 1986.

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Table 2. Chemical Shifts for Strongly Dipolar Shifted Residues in metCygba residue

Leu46(B10)e

proton

[B6]f

Phe49(B13)e [B9]f

Ala56(C4)e [C3]f Tyr59(C7)e [CD2]f

Phe60(CD1)e [CD3]f

Leu78(E4)e [E3]f

Ala82(E8)e [E7]f Arg84(E10)e [E9]f

Val85(E11)e [E10]f

NH CRH CβH’sb CγH CδH3’sb NH CRH CβH’s CδH’s CH’s CζH NH CRH CβH3 NH CRH CβH’sb CδH’s CH’s NH CRH CδH’s CH’s NH CR H CβH’sb CγH CδH3’sb NH CRH CβH3 NH CRH CβH’sb CγH’s NH CRH CβH CγH3’sb

δDSS(obsd)

8.44 4.12 2.45, 2.85 3.22 2.98, 1.75 8.14 4.30 3.18, 2.70 5.98 5.74 5.57 7.99 2.87 0.38 5.56 3.70 0.72, 1.77 6.27 6.11 7.46 4.37 7.39 8.83 8.17 5.31 1.45, 2.37 2.40 1.55, 0.82 11.07 5.48 2.44 8.42 3.08 -2.12(55),c 0.61 0.91, 1.30(?)d 8.93 1.62 2.79 0.38, 2.08(?)d

residue

Ala88(E14)e

[E13]f

Val109(F4)e [F11]f

Gly110(F5)e [F12]f Ala112 (F7)e [F14]f Ala114(F9)e [F16]f His117(FG3)e [FG2]f

Val119(FG5)e [FG4]f

Tyr123(G4)e [G2]f

Phe124(G5)e [G3]f

Leu127(G8)e [G6]f

proton

δDSS(obsd)

NH C RH CβH3 NH C RH CβH CγH3b NH CRH’sb NH C RH CβH3 NH C RH CβH3 NH C RH CβH’sb CδH CH NH C RH CβH CγH3’sb NH C RH CβH’sb CγH’s CζH’s NH C RH CβH’sb CδH’s CH’s CζH NH C RH CβH’sb CγH CδH3’s

6.88 3.79 0.52 8.24 6.13 3.47 4.02, 1.14 10.58 5.00, 5.95 9.34 5.16 2.95 9.99 4.60 1.11 7.30 4.57 2.08, 3.49 9.60 (20)c 6.57 8.08 2.60 1.48 -1.39(100),c -2.30(55)c 7.24 3.43 1.55, 1.35 6.56 6.50 7.92 2.94 3.27, 2.53 6.99 8.31 12.20 (20)c 7.65 2.92 0.27, -0.72 0.38 -0.55, 1.30(110)c

a Chemical shifts, in ppm, referenced to DSS, in 1H O, 50 mM in phosphate, pH 7.5 at 30 °C. b Diasterotropic protons (methyls) assigned stereospecifically 2 on the basis of selective NOESY cross peaks, differentiated relaxation or differentiated dipolar shifts, and are listed in order (i.e., Cβ1H, Cβ2H, Cγ1H3, Cγ2H3, c etc.). T1 values, in ms, for resolved residue protons are given in parentheses. d (?) indicates tentative assignment. e Helical/loop positions in mammalian (i.e., axial His is F8, distal is E7, etc.) Mb fold is given in parentheses. f Actual helical/loop positions in Cygb are given in square brackets.

strongly dipolar shifted residues are listed in Table 2, with the remaining data provided in the Supporting Information (Table S1). The helical fragment which contains the other axial His AMX spin system (as residue j + 3) exhibits the sequential NOESY cross peaks (Figure 5) represented by (Leu)j-(NR)j+1-(NR)j+2(AMX)j+3-Alaj+4-(NR)j+5-Zj+6-(Val/Thr)j+7, which the sequence identifies as the distal helical fragment Leu78-Val85 as residues E4-E11 on the distal or E helix. An additional fragment represented by Alak-Leuk+1(NR)k+2 must arise from the nearby distal helix fragment Ala88(E14)-Asn90(E16). The residues exhibit the expected11,12 NOESY contacts to the heme as depicted in Figure 1B. It was not possible to locate any other resolved and relaxed protons with NOESY connections to the His81(E7) backbone as would be expected for its ring NδH, and as is observed for His113(F8) in Figure 3F, or for the same distal His in metNgb.48 Distal helix residues, in addition to His81(E7), which exhibit significant dipolar shifts are Leu78(E4), Ala82(E8), Arg84(E10), and Val85(E11), whose shifts are listed in Table 2. The chemical shifts for His81(E7) are given in Table 1, and data for the remaining assigned residues are provided in the Supporting Information.

The two upfield hyperfine-shifted broad and strongly relaxed (T1 ≈ 3 ms) nonlabile proton signals (Figure 2A, 2B) can only arise from the ligated His four ring nonlabile protons.34,35,44 The closest protons to the CHs of each of the two His are the His CβH’s (to the ring CδH) and the ring NδH’s (to the ring CH). Saturation of the only clearly resolved peak at -9.5 ppm fails to exhibit (not shown) the sizable NOEs expected to His CβH’s if the peak arose from a CδH and, hence, identifies it as an axial His CH of one of the two His. The absence of a detectable NOE upon saturating this resolved His CH to the assigned His113(F8) NδH peak at 18.4 ppm argues for its origin as the His81(E7) CH. In view of the very similar hyperfine shift pattern of the two axial His backbones, it is most likely that the other upfield broad and strongly relaxed resonances under the 2-vinyl Hβ’s arise from the CH’s of His113(F8) (see below). The signals for the CδH’s of both axial His likely resonate in the broad composite peak (marked X) at ∼15 ppm in Figure 2B. Other Sequence-Specific Assignments. Backbone connections (Figure 5) to the assigned Ala114(F9) trace over the FG loop and identify Leu115(FG1), His117(FG3) in contact with pyrrole C (Figure 1B), Val119(FG5) in contact with the junction J. AM. CHEM. SOC.

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Figure 3. Portions of the 600 MHz 1H NMR NOESY spectrum of metCygb in 1H2O, 50 mM in phosphate, pH 7.5 at 25 °C (repetition rate 1 s-1, mixing time 60 ms), illustrating intra-heme key contacts: 1-CH3 to (E) 8-CH3, (A) 2-Hβs, (B) the δ-meso; 8-CH3 to (C) 7-propinate and the δ-meso; (A) intra-2vinyl and (B) 2-HR to the R-meso; (B, D) intra-6-propinate as well as (D) intra-His81(E7) and (D, E) intra-His113(F8) connections; His113(F8) to (D) Ala114(F9) and to (B) Val119(FG5) contacts. Also shown are contacts that establish heme orientation: (B) 1-CH3 to Val85(E11), Ala88(E14), and Val109(F4), 2-HR to Val85(E11); (C) 8-CH3 to Arg84(E10), Ala88(E14), and Val109(F4). The cross peak between His113(F8) NH and NδH in section F is shown with lower levels (by a factor of 1.5) of 60° shifted-sine-squared-bell apodization of the NOESY spectrum.

of pyrrole B and C (Figure 1B) and the backbone in Lys116(FG2) and Lys118(FG4). A helical section represented by ValmAMXm+1-AMXm+2-(NR)m+3-Ilem+4-Leum+5, with aromatic rings in contact with AMXm+1 and AMXm+2, uniquely identifies Val122-Leu127, residues G3-G8 on the G helix. The expected NOESY cross peaks to pyrroles A and B are observed for Tyr123(G4), Phe124(G5), and Leu127(G8) (Figure 1B), and the CζH of Phe124(G5) exhibits the strong relaxation (T1 ∼20 ms) predicted by the crystal structure (RFe ∼4.4 Å). Each of the residues exhibits significant dipolar shifts. A short helical sequence Alan-(NR)n+1-(NR)n+2-AMXn+3, the latter with contact to an aromatic ring, and with the Ala and the aromatic ring in contact with the junction of pyrroles B and C (Figure 1B), can only arise from Ala56(C4)-Tyr59(C7), with significant dipolar shifts exhibited by Ala56 and Tyr59. Backbone connections to Tyr59(C7) and a dipolar shifted, TOCSY-detected two-spin aromatic ring in contact with the pyrrole B/C junction (Figure 1B) identifies Phe60(CD1). The failure to detect either TOCSY or NOESY connections between 12994 J. AM. CHEM. SOC.

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the CH signal and CζH of the ring argues for a CζH shift that is essentially degenerate with that of CH’s. Two short helical fragments, neither with side chains exhibiting NOESY cross peaks to the heme, which are represented by Valp-(NR)p+1-Alap+2-(NR)p+3-AMXp+4-Alap+5 (with a four-spin aromatic ring in contact with AMXp+4) and Alaq-Zq+1-(NR)q+2Valq+3-(NR)q+4-AMXq+5-AMXq+6, must arise from Val27Ala32 and Ala44-Phe50 on helices A (A10-A15) and B (B8B14). Aromatic rings in contact with the backbone of residue q + 5 and q + 6 confirm Phe’s at positions B13 and B14. The residues exhibit the expected NOESY cross peaks to other residues, as shown in Figure 1B. Only Leu46(B10) and Phe49(B13) exhibit significant dipolar shifts. Contacts to Trp131(A12) and Phe49(B13) identify two additional G-helices residues Val130(G11) and Val135(G16). Resolution problems precluded unique identification of the H-helix, but expected (and observed) key contacts to the F and G helices (Figure 1B), together with unique TOCSY signals, identify Phe143(GH5), Trp151(H8), Val162(H19), Ala165(H22), Tyr166(H23), and Trp171(H28).

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Figure 4. Portion of the 500 MHz 1H NMR clean-TOCSY spectrum of metCygb in 1H2O, 50 mM in phosphate, pH 7.5 at 30 °C (repetition rate 1 s-1, mixing time 25 ms) illustrating scalar connections for the moderately relaxed CβH-CRH-NH of His81(E7), CRH-NH of His113(F8), Val109(F4), and the rings of Phe60(CD1) and Phe124(G5).

Figure 5. Fingerprint region of the 600 MHz 1H NMR NOESY spectrum of metCygb in 1H2O, 50 mM in phosphate at 25 °C (repetition rate 1 s-1, mixing time 60 ms), illustrating the characteristic helical Ni-Ni+1 connection for the (A, B) proximal helix (i ) F4-FG1 (Val109-Leu115); upper left of diagonal in (B), solid), and (B) the distal helix (i ) E4-E9 (Leu78-Cys83); lower right of diagonal, dash) and signature Ri-Ni+1 connection for (A, B) the proximal (solid) and distal (dash) residues.

Their position on the H-helix is confirmed by Ri-Ni+3/Ri-βi+3 contact involving Val162(H19), Tyr159(H16), and Ala165(H22). The chemical shifts for residues with significant dipolar shifts (g1 ppm for at least one proton) are listed in Table 2, and the remainder of the data are provided in the Supporting Information.

Orientation and Anisotropy of χ. The values of δdip(obsd) obtained via eq 4 for all assigned residues with significant temperature dependence to their chemical shifts were used as input in the five parameter searches for ∆χax, ∆χrh, R, β, and κ ) R + γ, using the two available sets of crystal coordinates for metCygb.11,12 In each case, very clear minima were observed J. AM. CHEM. SOC.

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Figure 6. Schematic depiction of the observed characteristic backbone NOESY connection that provided the assignment of seven helices and two interhelical loops. Helices and loops (the first and second lines) refer to those in vertebrate globins.

with excellent correlation between δdip(obsd) and δdip(calcd) for the predicted five parameters, as illustrated in Figure 7A and 7B. The values for the optimized parameters are compared in Table 3. The alternate crystal coordinates yield values for the five parameters for the alternate crystal coordinates that are well within their uncertainties. The plot of δdip(obsd) and δdip(calcd) with sequence number, shown in Figure 8A and 8B, respectively, attest to the excellent definition of the nodal pattern through the structure. Figure 8C plots the temperature gradient of the chemical shift, d[δDSS(obsd)]/d(T-1) versus sequence number. For the very few cases (marked by asterisk) where there appears to be a discrepancy between the signs of δdip(obsd) and δdip(calcd), the temperature gradient is found to be consistent with the sign of δdip(calcd), indicating that the minor discrepancies between δdip(calcd) and δdip(obsd) in Figure 8A and 8B arise from the uncertainty in the calculated values of δDSS(dia). Factoring the Dipolar and Contact Shifts. The δdip(calcd) for the heme and two axial His obtained by the alternate magnetic axes/anisotropies in Table 3 are listed in Table 1, where the experimental δhf can be factored via eq 6 to yield δcon for the heme methyl, meso-H’s, and the two axial His. It is noted that the δcon for the axial CβH’s and CH, which are dominated by the π-spin transfer into the highest bonding orbital 12996 J. AM. CHEM. SOC.

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of the imidazole ring, are indistinguishable for His81(E7) and His113(F8). Discussion

Active Site Solution Molecular Structure. The 1H NMR data reveal the presence of predominantly a single (J90%) molecular species. The fact that both Val119(FG5) and Phe60(CD1) make contact with pyrrole C (5-CH3) and that both Val85(E11) and Ala88(E14) make contact with the heme at the junction of pyrroles A and D dictates that the major isomer in solution has the heme orientation identified in the crystal structure, as depicted in Figure 1B, which is the same orientation as that in mammalian globins, but corresponds to the minor isomer orientation of metNgb.48 The two apparent heme methyl signals at 39.8 and 22.4 ppm in Figure 2A for a minor isomer (∼10%) were too weak to detect the NOESY contacts required for detailed structural characterization. However, the pattern of the methyl shifts is that expected (and for metNgb observed48) for the isoelectronic, six-coordinate complex with the hemin rotated by 180° about the R-,γ-meso axis relative to that of the major isomer. The sequence-specific assignment of both the His81(E7) and His113(F8) backbone in the major isomer, together with the expected and demonstrated moderate relaxation of the resolved Cβ2H signal for each ligand (as well as δcon; see

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NMR of Axial His Bonds in Cytoglobin

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Figure 7. Plot of δdip(obsd) versus δdip(calcd) for the optimized anisotropies and orientation of the paramagnetic susceptibility tensor χ when using the crystal coordinates of (A) C38S/C83S-metCygb: ∆χax ) 2.18 × 10-8 m3/ mol; ∆χrh ) -0.52 × 10-8 m3/mol; R ) 80°; β ) 6°; κ ) -4°, F/n ) 0.1011 and (B) WT metCygb: ∆χax ) 2.08 × 10-8 m3/mol; ∆χrh ) -0.49 × 10-8 m3/mol, R ) 120°, β ) 5°, κ ) 0°, F/n ) 0.1112 to evaluate the geometric factors in eq 2. Protons whose δdip(obsd) were used for the calculations are labeled in Table 1S (Supporting Information). Table 3. Anisotropies and Orientation of the Paramagnetic Susceptibility Tensor of metCygb C83S/C38S-metCygba

∆χ

ax

∆χrh R β κ

2.18 ( -0.52 ( 0.11c 80 ( 8° 6 ( 1° -4 ( 10° 0.08c

WT metCygbb

2.08 ( 0.08c -0.49 ( 0.11c 120 ( 8° 5 ( 1° 0 ( 10°

a Coordinates for chain B in ref 11. b Coordinates for metCygb in ref 12. c In units × 10-8 m3/mol.

below), directly confirms that the distal His81(E7) is ligated to the iron in solution. Both the patterns of heme-residue and interresidue NOESY cross peaks (summarized in Figure 1B) and the paramagnetically induced relaxation are consistent with the essentially indistinguishable active site molecular structure of the six-coordinated metCygb in the alternate crystal structures.11,12

The ability to detect a significant population of an equilibrium, five-coordinate isomer with a ruptured Fe-His(E7) bond, which would be high-spin and exhibit characteristic methyl signals in the extreme low-field (∼70-90 ppm) spectral window, depends on both the degree of population and the rate of the Fe-His(E7) bond scisson relative to the 1H NMR methyl chemical shift difference.50 We failed to detect by 1H NMR in solution any hyperfine shifted resonances characteristic of a five-coordinate, high-spin metCygb complex34 (if in slow exchange,50